Immortalized hepatocytes

ABSTRACT

The invention features a virally-immortalized mammalian hepatocyte, which is derived from a normal liver cell, has differentiated hepatocyte-specific metabolic activity, has the ability to proliferate, and is nontumorigenic after prolonged culture.

BACKGROUND OF THE INVENTION

The invention relates to immortalized hepatocyte cell lines.

Hybrid extracorporeal liver assist devices (LAD) are a promisingtherapeutic modality to reduce the potential of hepatic encephalopathy(HE) in patients with fulminant hepatic failure (FHF) (Sussman et al.,1995, Sci. Am. Science & Medicine May/June 1995:68-77). The exactidentity of the toxic metabolites responsible for HE are not clear, butbenzodiazepine-like compounds and ammonia have been implicated. Themetabolism of benzodiazepines in liver is accomplished by liver-specificcytochrome P450 enzymes.

Two cell based liver support systems have undergone clinical trials inhepatic failure patients. The first device (Sussman et al., 1994, Artif.Organs 18:390-396) contains the cell line C3A (Kelly, JH, W091/18087)which was subcloned from the hepatoma cell line Hep G2 (Knowles et al.,U.S. Pat. No. 4,393,133). The Hep G2/C3A cell line reportedly istumorigenic. The second device incorporates freshly isolated porcinehepatocytes (Rozga et al., 1994, Ann. Surg. 219: 538-546).

In a study comparing primary hepatocytes to HepG2 cells, the primarycells were superior to the transformed cell in every biotransformationpathway assessed (Nyberg et al., 1994, Ann. Surg. 220:59-67), butprimary hepatocytes only transiently provide metabolic functions. Manymetabolic activities are rapidly lost in culture.

SUMMARY OF THE INVENTION

The invention features virally-immortalized hepatocytes that retainliver-specific differentiated functions including, but not limited to,phase I oxidative enzymes involved in benzodiazepine metabolism. Suchclones can obviate the considerable expense and technical manpowercurrently required to obtain hepatocytes from large animals to seedliver assist devices. Additionally, these cells can be used in aconvenient in vitro method to test liver-specific characteristics aswell as to evaluate the toxicity of various compounds.

The invention features a virally-immortalized mammalian hepatocyte whichis derived from a normal liver cell, e.g., a porcine hepatocyte, hasdifferentiated hepatocyte-specific metabolic activity and the ability toproliferate, and is nontumorigenic after prolonged culture. By the term"virally-immortalized" is meant being transfected with all or part ofthe viral genome of a wild type or mutant virus. Preferably the virus isa DNA virus, more preferably the virus is simian virus 40 (SV40). By theterm "metabolic activity" is meant the ability to process a potentiallytoxic compound, e.g., a drug or endogenous metabolite, into a less toxicor non-toxic compound. By the term "normal liver cell" is meant a livercell which is not derived form a tumor. Preferably, the liver cell isnot derived from a transgenic animal. By the term "prolonged passage" ismeant greater than about 30 in vitro passages. Preferably thehepatocytes are nontumorigenic after 35 passages, more preferably after40 passages, more preferably after 45 passages, more preferably after 50passages, and most preferably after 60 in vitro passages.

The metabolic activity of the hepatocyte preferably includes P450 enzymeactivity. P450 enzyme activity includes the ability to metabolizediazepam, lidocaine, and/or 7-ethoxycoumarin (7-EC). The constitutivelevel of P450 enzyme activity is increased by contacting the hepatocytewith a metabolic inducer. By the term "metabolic inducer" is meant acompound which increases hepatocyte P450 enzyme activity at least 10%compared to the level of activity in the absence of the compound.Preferably, the metabolic inducer increases P450 activity by at least25%, more preferably by at least 50% compared to the level of activityin the absence of the compound.

The hepatocyte preferably contains a substantially pure SV40 DNA. Morepreferably, the SV40 DNA encodes the wild type SV40 large T antigen(TAg); most preferably, the DNA encodes the wild type TAg and does notencode other SV40 gene products. TAg expression may be constitutive orinducible. In the latter case, TAg-encoding DNA is operably linked to aninducible promoter, e.g., a dexamethasone (dex)-inducible promoter. TheDNA may also encode a temperature-sensitive TAg.

Hepatocytes containing DNA encoding constitutively expressed TAg andhepatocytes containing DNA encoding a temperature-sensitive TAg may alsocontain a substantially pure tumor suppressor-encoding DNA, e.g., DNAencoding human p53. In preferred embodiments, the substantially purehuman p53-encoding DNA is operably linked to a dex-inducible promoter.

"Substantially pure" as used herein refers to a DNA which has beenpurified from the sequences which flank it in a naturally occurringstate, i.e., a DNA fragment which has been removed from the sequenceswhich are normally adjacent to the fragment, e.g., the sequencesadjacent to the fragment in the genome in which it naturally occurs, andwhich has been substantially purified from other components whichnaturally accompany the DNA, e.g., DNA which has been purified from theproteins which naturally accompany it in the cell.

The invention also features a method of neutralizing a toxic compound,e.g., an endogenous compound such as a metabolite or anexogenously-administered compound such as a drug, in a bodily fluid of amammal which includes the step of contacting the bodily fluid, e.g.,blood or serum, with a virally-immortalized, nontumorigenic,metabolically-active hepatocyte. Preferably, the mammal is a human, morepreferably a human suffering from HE. The toxins in the fluid areefficiently processed by the hepatocytes, and thus, the fluid isrendered non-toxic. The processed bodily fluid may then be returned tothe patient from which it was derived. For example, the following toxinsmay be present in bodily fluid taken from a patient and neutralizedaccording to the invention: benzodiazepine, ammonia, 7-EC, lidocaine,and acetaminophen. Contact of the patient-derived bodily fluid with thehepatocytes may take place in any device capable of providing adequatecontact of isolated hepatocytes with a bodily fluid, such as a perfusiondevice, e.g., a LAD or bioreactor.

Virally-immortalized hepatocytes made according to the invention can betransferred to any type of perfusion device for use as the biologicalcomponent thereof.

The invention also includes a perfusion device which includes (a) ahousing defining a perfusion inlet and a perfusion outlet, (b) a porousmembrane structure mounted within the housing to define a perfusioncompartment and an adjacent hepatocyte compartment, and (c)virally-immortalized, nontumorigenic, metabolically-active mammalianhepatocytes.

Also within the invention is a method of evaluating the toxicity of acompound in vitro which includes the steps of (a) providing avirally-immortalized hepatocyte, (b) contacting the hepatocyte with thecompound, and (c) measuring the viability or metabolic activity of thehepatocyte. A decrease in viability or metabolic activity in thepresence of the compound compared to that in the absence of the compoundindicates that the compound is toxic or is likely to be toxic in vivo.

The invention also includes a method of evaluating the toxicity of ametabolite of a compound which includes the steps of (a) providing ametabolically-active hepatocyte, (b) contacting the metabolically-activehepatocyte with the compound to generate a cell supernatant which maycontain a toxic metabolite, (c) removing the cell supernatant from themetabolically-active hepatocyte, (d) providing a virally-immortalized,metabolically-active, nontumorigenic hepatocyte, (e) contacting theimmortalized hepatocyte with the supernatant, and (f) measuring theviability or metabolic activity of the immortalized hepatocyte. Thehepatocytes of step (a) may include any viable metabolically-activehepatocytes which may or may not be virally-immortalized. For example,if only a short term culture is required to generate the metabolite,primary hepatocytes may be used; if a long term culture is required,immortalized hepatocytes would be required. A decrease in viability ormetabolic activity in the presence of the supernatant compared to thatin the absence of the supernatant indicates that the compound is toxicor is likely to be toxic in vivo.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photograph of a Northern blot showing SV40 TAg genetranscription in subclone D63 in the absence of a metabolic inducer(lanes labeled "1") and in the presence of a metabolic inducer (laneslabeled "2").

FIG. 1B is a photograph of a Northern blot. As a control, the filtersshown in FIG. 1A were rehybridized with a glyceraldehyde-3-phosphatedehydrogenase (GAPDH) probe to assess loading differences.

FIG. 2 is a diagram of a perfusion device.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention provides immortalized hepatocyte cell lines, establishedby transfection with a viral transforming agent such as DNA encodingSV40 TAg. The cell lines are nontumorigenic and capable of persistentserial culture while maintaining differentiated liver-specific metabolicactivity. Unlike the anchorage-dependent normal primary porcinehepatocytes from which they were derived, these cells require nospecific substrates for adhesion and growth. In addition, serum is notnecessary either for proliferation or for maintenance of differentiatedfunction.

The cell lines of the invention are classified into five categories.Line I is characterized by constitutive expression of wild type SV40TAg. Following transfection with wild type SV40 TAg-encoding DNA,transfectants were screened for the ability to proliferate in culture(express SV40 TAg), maintain metabolic activity, in particular theexpression of P450 metabolic enzymes such as those required tometabolize diazepam, and maintain other hepatocyte-specific phenotypiccharacteristics, e.g., cell morphology and expression ofhepatocyte-specific cytoskeletal or enzymatic markers. Cell lines canalso be screened for other differentiated liver-specific enzymaticactivities, e.g., glucuronidation metabolic activity and the ability tometabolize ammonia or other toxic compounds.

In addition to Line I, four other classes of virally-immortalizedhepatocyte cell lines (Lines II, III, IV, and V) were developed. Tofurther ensure safety in clinical applications, e.g., human therapeuticuse such as cell transplantation, Lines III, IV, and V have been furthermodified to prevent undesirable proliferation, e.g, tumorigenicity.Proliferative activity and/or metabolic activity are regulatable in eachof these cell lines. Regulatable proliferative activity contributes tothe safety of these cells lines. For example, Line III is characterizedby thermally-regulated (mutant) SV40 TAg expression; Line IV ischaracterized by constitutively expressed SV40 TAg with dex-induciblep53 expression; and Line V is characterized by thermally-regulated SV40TAg expression with dex-inducible p53 expression.

Line II which is suitable for in vitro diagnostic use and toxicologytesting is characterized by dex-inducible wild type SV40 TAg expression.

Reagents

The following reagents were used in establishment and culture ofimmortalized hepatocytes:

Chee's essential media (CEM), gentamicin, and fetal bovine serum (FBS)were purchased from GIBCO (Grand Island, N.Y.); insulin was purchasedfrom Eli Lilly (Indianapolis, Ind.); and dex was purchased fromElkins-Sinn (Cherry Hill, N.J.). Vitrogen was obtained from the CollagenCorporation (Palo Alto, Calif.); collagenase from WorthingtonBiochemical Corporation (Freehold, N.J.). All other chemicals wereobtained from Sigma Chemical Co. (St. Louis, Mo.), Aldrich Chemical(Milwaukee, Wis.) or J.T. Baker (Medford, Mass.) unless otherwise noted.Acetaminophen metabolites were obtained from McNeil Pharmaceuticals(Fort Worth, Pa.).

Establishment of immortalized hepatocytes

Five cell lines were developed. Normal primary porcine hepatocytes weretransfected with plasmids encoding SV40 TAg (wild type ortemperature-sensitive mutant) and the bacterial gene encoding neomycinresistance. SV40 DNA was integrated into the nuclear DNA of the primaryporcine hepatoctyes. In some cases, cells were secondarily transfectedwith DNA encoding a tumor suppressor. Using a lipofection technique,transfection efficiency in porcine hepatocytes was greater than 5%.

For Line I, the cells were transfected with wild-type TAg expressed froma constitutively active promoter/enhancer (SV40 early promoter).Sixty-four initial clones were identified by their ability toproliferate in culture, i.e., form colonies. Twenty-four of those cloneswere further studied. Twenty-four clones maintained typical hepatocytemorphology and the ability to proliferate in culture. Twenty-three outof twenty-four clones maintained metabolic activity, e.g., the abilityto metabolize diazepam. Clone D63 was subcloned based on high P450metabolic activity as determined by fluorescence activated cell sorting(FACS) analysis. Five subclones (D63A, D63F, D63G, D63H, and D63I) wereidentified as having high P450 activity.

The cells of Line I showed no evidence of tumorigenicity. Cells wereinjected into immunocompromised (severe combined immunodeficient (SCID))mice after 12 passages (P-12); the cells failed to form tumors. Incontrast, HepG2 cells under similar conditions formed tumors.Chromosomal analysis revealed that subclone D63H had 35-39 chromosomesafter passage 8 (P-8) and 30-38 chromosomes after passage 31 (P-31);normal pig cells have a diploid chromosome number of 38. No grossstructural abnormalities were observed. These cells also maintained arange of hepatocyte specific functions and displayed typical hepatocyteintracellular morphology.

Despite their lack of tumorigenicity, an additional mechanism to preventtumor formation was introduced. For Line IV, a portion of cells fromLine I were secondarily transfected with an inducible gene to halt cellproliferation. Highly differentiated clones were selected andtransfected with a dex-inducible wild type human p53 gene. Integrationof the p53 gene provides a regulatable "on-off" mechanism to controlcell proliferation. Because dex induces p53 expression in these cells,any clinical application, e.g., use of an LAD or cell transplantation,that exposes these cells to human blood or serum would (by the presenceof dex) prevent proliferative activity.

In Line II, wild type SV40 TAg-encoding DNA is fused to an induciblepromoter, mouse mammary tumor virus (MMTV), which favors expression ofTAg only in the presence of the inducer, dex. The transfecting plasmidfor this line included a MMTV promoter fused to the TAg gene. In thiscell line, TAg gene expression (indicative of cell proliferation) isregulated by varying the amount of dex available to the cells. Aregulatable transforming gene allows adjustment of the intracellularconcentration of the transforming protein. Thus, the cell's ability toproliferate and its differentiation potential can be controlled. Forexample, in the presence of dex, the cells proliferate, but in theabsence of dex, proliferation is reduced or halted and the cells expressdifferentiated liver-specific metabolic activity.

Line III was created by transfection of normal primary porcinehepatoctyes with a temperature-sensitive TAg gene. For the establishmentof Line V, selected clones from Line III were secondarily transfectedwith the wild type human p53 gene. In both Lines IV and V, p53suppresses TAg. The p53 nucleoprotein gene product inactivates TAg byforming an oligomeric complex with it. Thus, the concentration ofintracellular active TAg is manipulated by increasing the relativeproportion of inactive (p53-complexed) TAg.

Plasmids

Line I which constitutively expresses wild type SV40 TAg was created bytransfecting primary hepatocytes with DNA encoding wild type SV40 TAg(Fiers et al., 1978, Nature 273:113-120, hereby incorporated byreference). The source of the wild type TAg was pBR/SV (ATCC #45019).The pBR/SV plasmid contains the entire SV40 genome inserted in the BamHIsite of pBR322. The initial construct containing a KpnI-BamHI fragmentfrom pBR/SV encompassing the SV40 promoter and the entire early region,was inserted into KpnI and Bam HI-digested Bluescript S-K (Stratagene,La Jolla, Calif.). The resulting construct, Blue-TAg, contains the SV40promoter-T antigen fragment.

Line II which inducibly expresses SV40 TAg was created by transfectingprimary hepatocytes with DNA encoding wild type SV40 TAg under thecontrol of the dex-inducible MMTV promoter. The AvrII-BamHI fragment ofSV40, containing the early region and lacking the viral promoter, wasblunt-ended and ligated into SalI-digested, blunt-ended pMAMneo. Thelatter contains a hybrid long terminal repeat (LTR) which contains theRous Sarcoma Virus (RSV) enhancer and dex-inducible MMTV promoter.Correct orientation of the insert in the resulting plasmid,pMAMneo-SV_(T), was confirmed by restriction analysis.

Line III which expresses a temperature-sensitive SV40 TAg was created bytransfecting primary hepatocytes with DNA encoding a mutant SV40 TAg(SV40^(ts) A58). DNA encoding the temperature-sensitive SV40 TAg has amutation within the SV40 A gene (Jat et al., 1989, Mol. Cell Biol.9:1672-1681). At 33° C., the cells proliferate and express SV40 TAg buthave low liver-specific differentiated functions. At 39° C., the cellscease proliferation and express liver-specific functions such asmetabolic activity.

Lines IV and V were created by secondarily transfecting cells from LinesI and III respectively with DNA encoding the tumor suppressor, p53. Thegene encoding human p53 (tumor suppressor) was obtained from theAmerican Type Culture Collection (ATCC) as a 2 kb cDNA insert in pBR322(php53B; ATCC #57254). The fragment, between Nhel and SalI restrictionenzyme sites which contains wild type p53 gene from php53B was insertedinto Nhel-SalI digested pMAM resulting a new construct, pMAM-p53.

Transfection Method

Porcine hepatocytes were isolated and established in culture by knownmethods. Transfection of normal primary porcine hepatocytes was carriedout as follows. Freshly isolated porcine hepatocytes were seeded 2×10⁶cells/dish and maintained in standard CEM medium supplemented with 10%FBS (10% FBS-CEM) overnight (1-day culture). The 10% FBS-CEM media ofthe 1-day cultures were replaced with 5% FBS-CEM. For Line I, Blue TAg(6.4 μg) plus pRSVneo (1.6 μg) (molar ratio 5:1) were dissolved in 200μl OPTI-MEM (GIBCO/BRL, Gaithersburg, Md.). For Line II, the plasmidpMAMneo/SV_(T) (5 μg) was dissolved in 200 μl OPTI-MEM (GIBCO/BRL). ForLine III, the plasmid encoding temperature-sensitive SV40 TAg (9 μg)plus 1 μg of pRSVneo (molar ratio 5:1) were dissolved in 200 μl ofOPTI-MEM (GIBCO/BRL). The plasmid-medium from Lines I-II was thencombined with 200 μl OPTI-MEM which contained 20 μl of LIPOFECTIN®reagent (GIBCO/BRL) and incubated at room temperature for 20 min.Following incubation, this mixture was gently added to the porcinehepatocyte cultures.

The transfectants of Lines I & II were incubated for 6 hours at 37° C.,and the transfectants of Line III were incubated for 6 hours at 33° C.(the temperature at which the cells proliferate). The DNA-containingmedium was replaced with 10% FBS-CEM, and the transfectants werecultured for 5 days, during which the media was replaced every 48 hours.

Selection of transfected cells

Successfully transfected cells contained both TAg and the bacterialneomycin-kanamycin resistance gene, neo. The addition of G418(GIBCO/BRL) to the culture medium killed any cells which had notintegrated the neo gene within 10 days. Cells which contained the neogene (but not TAg-encoding DNA) died as is typical of primary culturedcells.

Transfected cells were selected by the addition of G418 (12.5 mg/mlmedia). After G418 selection was terminated, the transfected cells grewand formed colonies. Single clones were transferred to fresh dishes andmaintained in culture.

Successful transfection of the cells with TAg-encoding DNA was confirmedby standard Northern blot analysis to detect the presence of SV40 TAgmRNA.

Secondary transfection

Subconfluent cultures of cells from Line I or III derived from an earlypassage were subjected to a secondary transfection. The medium of theculture of Line I or III was replaced with 5% FBS-CEM. PlasmidpMAMneo/p53 (10 μg) was added to 200 μl OPTI-MEM, and LIPOFECTIN®reagent (20 μl) was added to 200 μl OPTI-MEM. The plasmid andlipofection reagent solutions were then combined and incubated at roomtemperature for 20 min. Following incubation, a 200 μl aliquot was addedto each culture plate of line I or III. For Line I, cultures wereincubated at 37° C. for 6 hours, and for Line III, they were incubatedat 33° C. for 6 hours. The medium of each of the cultures was thenreplaced with 10% FBS-CEM. The cells of Line I were cultured at 37° C.for 3 days, and the cells of Line III were incubated at 33° C. for 3days. After the 3-day culture, the cells were divided yieldingapproximately 1×10⁶ cells per culture plate. For Line I, the cells wereincubated for 7-10 days in modified CEM media (dex-free) at 37° C., andfor Line III, the cells were incubated for 7-10 days in modified CEMmedia at 33° C. Single clones were transferred to individual dishes.

Immortalized cells can be further transfected with additional genes,e.g., those encoding absent or defective gene products, prior to use asa human therapeutic in order to compensate for absent or defective geneproducts in a human patient.

Selection of cells with P450 enzyme activity

The formation of fluorescent products from a non-fluorescent substrate,5.6-methoxycarbonylfluorescein, by hepatocytes via the P450 metabolicpathway was used as the basis for FACS to identify and concentrate apopulation of immortalized hepatocytes with high P450 enzyme activity.

Hepatocytes were labeled with 5.6-methoxycarbonylfluorescein as follows.Cultured cells were washed with phosphate buffered saline-pH 7.4 (PBS)and plated on tissue culture plates. The substrate solution was preparedby adding 5.6-methoxycarbonylfluorescein (Molecular Probes, Eugene,Ore.), to PBS to a final concentration of 100 μM/L. The substratesolution (5 ml) was added to the washed plated cells. The platescontaining cells and substrate solution were incubated in darkness at37° C. for 15 min. and then washed with PBS. For FACS analysis, thecells were removed from the culture plates by trypsin digestion andresuspended in 10% FBS-CEM supplemented with antibiotics. The cellsuspension was then subjected to FACS analysis or stored at 4° C. untilFACS analysis.

FACS analysis was performed using the Becton Dickinson (Rutherford,N.J.) FACSort machine. The 486 nm line of an argon laser was used forfluorescence excitation. Emission was measured using a 520-560 nmbandpass filter. Fluorescent cells were deemed to be positive for P450activity. The brightest fluorescent cells (e.g., the top 5% of thecells) were sorted into 50 ml tubes containing medium. Weaklyfluorescent cells were discarded.

Cells with P450 activity were centrifuged for 20 min. at 7×g,resuspended in medium, and plated in tissue culture dishes.

Immunofluorescent staining

To characterize the cell type of the immortalized cells,immunofluorescent staining was carried out using anti-keratin-8 andanti-keratin-18 antibodies. These antibodies bind to hepatocytes but notother liver cells, such as fibroblasts, Ito cells, smooth muscle cellsand bile duct cells.

A cell sample was fixed with absolute acetone and then stained withhepatocyte-specific antibodies as follows. The cells were cytospun ontopretreated slides (Superfrost/plus, Fisher Scientific, Pittsburgh, Pa.)and incubated with mouse anti-keratin-8 and mouse anti-keratin-18(Amersham, Arlington Heights, Ill.) for 30 min. at 37° C. After rinsingwith PBS, bound immunoglobulin (IgG) was detected by further incubationfor 30 min. with fluorescein isothiocyanate (FITC)-conjugated goatglobulins directed against mouse IgG (Sigma, St. Louis, Mo.). The slideswere stained with propidium iodide (Sigma) to show nuclei.

Evaluation of metabolic activity

P450 metabolic activity (e.g., diazepam metabolism, lidocainemetabolism, 7-EC metabolism and dealkylase activity) and glucuronidationactivity (e.g., acetaminophen metabolism) were analyzed as follows.Substrates were added to hepatocyte cultures (diazepam (50 μg/ml); 7-EC(50 μg/ml); and acetaminophen (5 mM; 0.75 mg/ml) and incubated for 3hours prior to evaluating viability and/or metabolic activity. A mediacontrol was incubated simultaneously under similar conditions in theabsence of cells. Thymidine incorporation by cultured hepatocytes wasmeasured to assess cell proliferation in vitro. Media from cellcultures, i.e., culture supernatants, were collected and stored at -30°C. until assayed. After removal of the culture supernatants, the cultureplates were rinsed 3 times with phosphate buffered saline (PBS) andreserved for protein determination by known methods, e.g., Hayner et al.1982, Tissue Culture Methods 7:77-80.

Diazepam metabolic activity was measured as follows. 4×10⁶ cells werecultured in a monolayer in 5 ml of medium containing 50 μg/ml diazepam,and the amount of diazepam metabolites present in the culturesupernatant measured after 3 hours of culture. Diazepam metabolites wereassayed by high performance liquid chromatography (HPLC) using a C18μ-Bondpack reverse phase column according to known methods, e.g.,Jauregui et al., 1991, Xenobiotica 21:1091-1106.

7-EC metabolism was measured as follows. 4×10⁶ cells were cultured in amonolayer in 5 ml of medium containing 50 μg/ml 7-EC, and the amount of7-hydroxycoumarin present in the culture supernatant measured after 3hours of culture. 7-hydroxycoumarin levels were assayed using a C18p-Bondpack reverse phase HPLC column according to known methods, e.g.,Jauregui et al., 1991, Xenobiotica 21:1091-1106.

Acetaminophen and its metabolites were determined by ion-pairing HPLCusing a C18 reverse phase column. Acetaminophen metabolism was measuredas follows. 4×10⁶ cells were cultured in a monolayer in 5 ml of mediumcontaining 5 mM acetaminophen (0.756 mg/ml), and the amount ofacetaminophen glucuronide present in the culture supernatant measuredafter 3 hours of culture. Acetaminophen and its metabolites, e.g.,acetaminophen glucuronide, were determined by ion-pairing highperformance liquid chromatography, e.g, using the method of Colin etal., 1986, J. Chromatogr. 377:243-251. Acetaminophen may also bemetabolized via a sulfonation pathway; metabolites may be assayed usingmethods known in the art.

Lidocaine metabolism is measured using known methods, e.g., Jauregui etal., 1995, Hepatology 21:460-469. For example, 4×10⁶ cells were culturedin a monolayer in 5 ml of medium containing 20 μg/ml lidocaine, and theamount of lidocaine metabolite, e.g., monoethylglycinexylidide (MEGX)present in the culture supernatant is measured after 3 hours of culture.Metabolism of lidocaine is tested using a TDX Analyzer manufactured byAbbott Diagnostics Laboratories, No. Chicago, Ill.

Ammonia metabolism is also measured according to methods known in theart, e.g., using the commercial analyzer, Ektachem, manufactured byKodak Corp. Rochester, N.Y. Ammonia metabolism is detected by measuringthe amount of ammonia remaining in the culture supernatant after 3 hoursof culture.

Characterization of immortalized hepatocytes

Normal primary porcine hepatocytes were transfected with SV40 DNA tocreate immortalized cells. Stable cell lines were selected andmaintained for more than 40 passages. Immortalized hepatocytes maintaindifferentiated liver-specific functions such as metabolic activity, inparticular P450 enzyme activity (e.g, diazepam metabolism (TABLE 2),lidocaine metabolism, 7-EC metabolism (TABLE 3), and dealkylase activity(TABLE 1)) and glucuronidation activity (e.g., acetaminophen metabolism(TABLE 4)).

                  TABLE 1    ______________________________________    Fluoresceins of mixed function oxidase in    porcine hepatocytes D clone.    ______________________________________    D1          ++         D11#1      ++++    D2          ++         D11#2      ++++    D3          +          D41        +++    D4          ±       D46        ++++    D6          +++        D54        ++    D7          +          D61        ++    D8          ±       D63        ++++    D9          ++         D64        +    D10         ±       D58        ±    D12         ++    D13         ±    D14         ++    D15         ++    D16         ±    ______________________________________     5.6, methoxycarbonylfluorescein is a substrate of p450 dealkylase

                  TABLE 2    ______________________________________    Diazepam Metabolic Activity of Porcine Hepatocyte Clones    (μg total diazepam metabolites/mg cell protein)    Passage          D 63 A  D 63 F  D 63 G                                D 63 H                                      D 63 I                                            Notes    ______________________________________    6     19.71   23.95   4.46  5.41  15.31    7     12.66   13.94   12.59 4.59  17.52    9     22.13   20.38   18.42 7.59  22.21    11    22.56   30.54   26.84 8.78  36.84    12    1.39    1.61    1.74  0.00  1.78  #1) 10% FBS                                            only    "     10.37   9.60    11.22 3.80  11.99 #2) 10% FBS +                                            rifampin                                            (non-coated                                            plates)    "     12.86   13.53   12.56 4.51  12.30 #3) 10% FBS +                                            rifampin    13    3.60    7.42    0.00  --    3.77  #1) Rifampin +                                            0% FBS    "     11.86   11.31   7.24  --    7.04  #2) Rifampin +                                            10% FBS    "     0.00    0.00    0.00  --    0.00  #3) 0% FBS                                            only    "     0.85    0.83    1.22  --    0.92  #4) 10% FBS                                            only    "     2.68    4.23    0.00  --    0.00  #5) Rifampin +                                            0% FBS                                            (non-coated                                            plates)    "     10.82   9.90    7.71  --    6.22  #6) Rifampin +                                            10% FBS                                            (non-coated                                            plates)    15    8.51    12.88   4.57  0.00  4.35    17    14.78   12.87   8.88  15.63 10.48    18    13.15   6.63    20.63 16.59 3.72    19    16.56   13.35   16.40 16.26 10.74    20    26.88   21.06   21.58 37.72 27.26    21    5.30    19.26   10.51 18.03 11.36    22    0.00    10.53   6.69  31.54 4.78    23    0.00    6.56    5.01  13.05 0.00    24    4.67    5.51    7.99  25.95 6.78    25    0.00    14.16   17.80 41.44 0.00    26    0.00    6.24    0.00  16.07 7.74    27    0.00    5.86    0.0/1.68                                0.00  0.00                          (6-1)    30    0.00    6.89    10.12 26.39 27.43    31    0.00    7.93    7.67  16.47 32.15    32    --      16.42   13.86 --    24.53    33    --      --      --    31.22 --    33    --      1.70    2.25  30.09 0.00    34    0.00    15.52   8.84  19.40 26.99          (P-33)    35    --      2.63    --    --    10.87    --    --      --      3.15  30.36 --                          (P-34)                                (P-36)    --    --      5.19    5.56  48.20 15.12                  (P-36)  (P-35)                                (P-37)                                      (P-36)    --    --      14.24   7.74  12.80 27.23                  (P-37)  (P-36)                                (P-38)                                      (P-37)    --    0.00    11.00   9.67  26.90 25.38          (P-36)  (P-38)  (P-37)                                (P-39)                                      (P-38)    --    0.00    14.90   10.23 8.97  23.71          (P-38)  (P-42)  (P-41)                                (P-43)                                      (P-42)    ______________________________________

                  TABLE 3    ______________________________________    Metabolism of 7-Ethoxycoumarin in Porcine Hepatocyte Clones    Seeded on Lux Permanox Dishes             7-Hydroxycoumarin                           T-Protein    Sample   (μg/ml)    (mg)      μg/mg protein    ______________________________________    EXPERIMENT #1    D 63 F P-42             0.58          3.55      0.82    D 63 G P-41             0.91          3.63      1.25    D 63 I P-42             1.69          3.70      2.28    EXPERIMENT #2    D 63 H P-43             0.38          3.23      0.59    ______________________________________     *Clones were incubated with 50 μM 3methylcholanthrene for 72 hours     prior to 7EC incubation

                  TABLE 4    ______________________________________    Acetaminophen Glucuronide Production in Porcine    Hepatocyte Clones Seeded on Lux Permanox Dishes    ______________________________________    EXPERIMENT #1    Sample     μg/ml Acetaminophen glucuronide    ______________________________________    D 11 #1 P-8               5.8    D 11 #2 P-8               2.8    D 41 P-8   8.7    D 46 P-8   No drug in sample    D 54 P-8   11.4    D 61 P-8   3.4    D 64 P-8   3.1    ______________________________________    EXPERIMENT #2    Sample     Acetaminophen glucuronide μg/ml    ______________________________________    D 63 P-8   5.7    ______________________________________              Acetaminophen              glucuronide  T. Protein    Sample    (μg/ml)   (mg)     μg/mg protein    ______________________________________    EXPERIMENT #3    D 63 F P-42              20.28        3.78     26.83    D 63 G P-41              24.88        3.54     35.14    D 63 I P-42              31.45        3.70     42.50    EXPERIMENT #4    D 63 H P-43              37.38        3.53     52.95    ______________________________________

At low plating density (less than 0.5×10⁶ cells per 21 cm² dish), thecells proliferate and exhibit low levels of differentiatedliver-specific functions. However, as the cultures reach confluence(e.g., at 50% confluence), the cells express differentiatedliver-specific functions and become responsive to metabolic inducers.P450 metabolic activity is constitutively expressed and induced to ahigher level of expression and/or activity by a variety of metabolicinducers. Metabolic inducers include polycyclic aromatic hydrocarbons(e.g., benzo a!pyrene, 2,3,7,8-tetrachlorbenzo-p-dioxin; TCDD),isosafrole, omeprazole, phenobarbitol, methylcholanthrene, alcohol,acetone, pyrazole, imidazole, rifampin, glucocorticoids, miconazole,erythromycin and other macrolide antibiotics, peroxisome proliferators(e.g., clofibrate, phthalate esters, and halogenated hydrocarbonsolvents). For example, metabolic inducers, e.g., rifampin,phenobarbitol and methylcholanthrene, introduced into a low densityculture of immortalized cells inhibited proliferation and actuallyincreased cell death. When the same metabolic inducer was introduced tocells cultured at higher density, the cells showed increased metabolicactivity without significant cell death. The cell lines have been grownin quantity in flasks and roller bottles with or without microcarriersor protein substrates (TABLE 5).

The immortalized cell lines also retain many other characteristics ofnormal primary hepatocytes. Immunocytochemical staining revealed thepresence of carbamyl phosphate synthetase, an early enzyme of the ureacycle. The immortalized cells also stained positively for cytokeratin 8and cytokeratin 18, hepatocyte-specific cytoskeletal proteins. Electronmicroscopic evaluation revealed that the cells retained morphologiccharacteristics of normal hepatocytes. These data indicate that theimmortalized cells retain hepatocyte-specific phenotypic markers.

The cells were also stained for the presence of SV40 TAg. Each of thecell lines stained positively for SV40 TAg confirming the integration ofSV40 DNA into nuclear DNA of the immortalized cell. Northern blotanalysis confirmed the presence of TAg mRNA (FIGS. 1A and 1B).

Use

The immortalized cells metabolize substances implicated in HE (an oftenfatal complication of acute hepatic failure). These cells are thereforeuseful as the biological component of a LAD.

The cell lines can also be used in (i) intra-corporeal hepatic therapy(cell transplantation), (ii) in vitro toxicology testing, and (iii)hepatocyte function studies. Cell lines I, III, IV and V are suitableand safe for clinical applications because their proliferation can becontrolled, i.e., halted, in clinical in vivo settings. Cell line II issuitable for in vitro diagnostic assays and toxicology testing.

Structure of Perfusion Device

FIG. 2 shows a perfusion device. The device includes a rigid, plasticouter shell 12, a plurality of hollow semi-permeable membrane fibers 14therein, and outer caps, 16 and 18. Fibers 14 are porous fibers. Theupper and lower ends of hollow fibers 14 are potted in potting material15 and thereby sealed to the inner surface of shell 12 near the upperand lower ends, employing techniques which are well known in the art.Cap 16 has perfusion inlet 20, and cap 18 has perfusion outlet 22, bothof which communicate with the interiors of hollow fibers 14. Ports 24and 26 are inward of potting 15 and provide access to the region withincontainer 12 external of hollow fibers 14. Fibers 14 act as a barrierbetween perfusion compartment 25, inside of the fibers 14 and hepatocytecompartment 27, in the region between the exterior surfaces of fibers 14and the inside of shell 12.

Artificial liver 10 is made from a standard shell provided with pottedhollow fibers according to procedures well known in the art. Fibers 14are made of membranes which include but are not limited to polyacrylicpolyurethane, cellulose acetate or polysulfone polymer, have outerdiameters between 150 μm and 400 μm, have inner diameters between 50 μmand 350 μm, and have pores of a size to have molecular weight cutoffs of40,000 to 250,000 daltons. The outer surfaces of fibers 14 may betreated, e.g., by treatment of the fibers with collagen, lectin,laminin, or fibronectin, or left untreated for the attachment ofimmortalized cells. The perfusion device may be regenerated by removingthe hepatocytes from the hepatocyte compartment and replenishing thecompartment with a fresh aliquot of immortalized hepatocytes.

Toxicology Testing

Immortalized hepatocytes may also be used as a screening tool toevaluate the toxicity of a compound, e.g., a drug to be administered toa patient or a metabolite of such a drug. The in vitro assay includesthe following steps: providing a sample of immortalized hepatocytes;contacting the sample with the compound to be tested, e.g., for at least15 min.; and measuring the viability of the sample. Some compounds mayrequire contacting the hepatocytes for longer than 15 min., e.g., 1hour, 3 hours, 24 hours, or up to several days, in order to determinethe effect of the compound on the hepatocytes. Viability of the cellsmay be measured by staining the cells with a vital dye such as trypanblue, or detecting lactate dehydrogenase (LDH) leakage. Alternatively,propidium iodide staining can be used to assess cell viability. Adecrease in viability of the hepatocytes in the presence of the testcompound compared to that in the absence of the same compound indicatesthat the compound is or is likely to be toxic in vivo. Alternatively,rather than measuring cell viability following contact with the testcompound, metabolic activity of the cells may be measured. In this case,a decrease in metabolic activity, e.g, the ability to metabolizediazepam, in the presence of the test compound compared to that in theabsence of compound indicates that the compound impairs the enzymaticfunction of the liver, and therefore, is or is likely to be toxic invivo.

Some compounds, e.g., drugs administered to a patient, are not toxicupon administration but are subsequently metabolized into a toxicmetabolite, which in turn may impair the function of liver cells orother cells and/or cause cell death. Thus, the invention, includes an invitro toxicology assay to evaluate the toxicity of metabolites of testcompounds. The method includes the steps of: providing ametabolically-active hepatocyte; contacting the metabolically-activehepatocyte with a test compound to generate a cell supernatant; removingthe cell supernatant from the metabolically-active hepatocyte; providinga virally-immortalized, metabolically-active, nontumorigenic hepatocyte;contacting the virally-immortalized hepatocyte with the supernatant; andmeasuring the viability of the immortalized hepatocytes. As above, somecompounds may require contacting the hepatocytes with the compound orsupernatant, respectively, for longer than 15 min., e.g., 1 hour, 3hours, 24 hours, or up to several days, in order to generate a toxicmetabolite or to determine the effect of the compound on the indicatorhepatocytes, respectively. Any viable metabolically-active hepatocytesmay be used to generate the cell supernatant. For example, primaryhepatocytes can be used if only a short term culture is required togenerated the metabolite to be tested. For longer culture times, e.g.,several days, virally-immortalized hepatocytes are used.Virally-immortallized hepatocytes are the indicator hepatocytes, i.e,the hepatocytes which will be evaluated for viability and/or metabolicactivity. A decrease in viability of the indicator hepatocytes in thepresence of the supernatant compared to that in the absence of thesupernatant indicates that the test compound is or is likely to be toxicin vivo. Alternatively, rather than measuring the viability of theindicator hepatocytes, their metabolic activity can be measured asdescribed above. In this case, a decrease in the level of metabolicactivity in the presence of the supernatant compared to that in theabsence of the supernatant indicates that the test compound is or islikely to be toxic in vivo.

Cell transplantation

Immortalized cells can be transplanted into individuals in need of liversupport functions, e.g., those suffering from HE. For example, tominimize transplant rejection, the cells can be encapsulated in amembrane which permits exchange of fluids but prevents cell/cellcontact. Transplantation of microencapsulated hepatocytes is known inthe art, e.g., Balladur et al., 1995, Surgery 117:189-194; and Dixit etal., 1992, Cell Transplantation 1:275-279.

Immortalized hepatocytes may also be transfected with DNA encoding ahuman gene product which is absent or defective using known methods,e.g., Raper et al., 1993, Transplantation 2:381-400. Such cells may betransplanted into the affected individual to produce the recombinantgene product in vivo.

Advantages

The immortalized hepatocytes described are unique in that they maintaindifferentiated liver-specific metabolic activity concurrent withproliferative activity. Typically, as differentiation progresses,proliferation diminishes (Freshney, R.I., 1987, Culture of animal cells:A manual of basic technique, 2nd ed., New York, N.Y., Alan R. Liss, Inc.p. 187). Except in tumor cells, long-term maintenance of these functionsare incompatible. The immortalized hepatocyte cell lines of theinvention exhibit a range of liver-specific functions and arenontumorigenic when injected into SCID mice.

A major advantage of the cell lines of the invention is their lack oftumorigenicity, even after prolonged culture. A number of additionalsafety measures have been engineered into nontumorigenic cells of Line Iin order to make them even safer for clinical use, particularly in vivotherapeutic use. For example, Line III expresses a temperature-sensitiveSV40 TAg which is non-functional at physiologic temperatures, and LinesIV and V express a dex-inducible tumor suppressor, human p53.

The cells can be produced in quantities adequate for commercial usageand are not dependent on extracellular matrix proteins or othersubstrates, e.g., collagen, lectin, laminin, or fibronectin, for surfaceadhesion or activity (TABLE 5) which allows a greater variety ofbiomaterials to be considered for LAD design. The need for collagenaseis obviated. The reduced need for serum supplementation also decreasescommercial costs.

                  TABLE 5    ______________________________________    Diazepam Metabolite Production                 Vitrogen Coated                           Non-Coated    ______________________________________                   15.2        14.1    CEM + 2% FBS   19.5        16.1    CEM + 5% FBS   21.17       19.3    CEM + 10% FBS  21.1        19.4    ______________________________________

Freshly isolated hepatocytes and hepatoma-derived cells have been usedas the biological component of LADs. Neoplastic cell lines may notprovide all necessary metabolic functions and are potentially risky touse in devices that are in contact with the patient's bloodstream.However, the provision of a continuous supply of primary or freshlyisolated hepatocytes from large animals to seed LAD involves expensiveprocedures and maintenance of large animal colonies. Freshly isolatedporcine hepatocytes have been shown to have greater biotransformationcapacity than the HepG2/C3A cell line, but the use of fresh primarycells presents logistical problems (i.e., on call surgical personnel andfacilities, animal access and transportation/storage needs). The celllines described herein have equivalent or higher P450 activity thancryopreserved primary cells. For example, both cell of Line I and freshhepatocytes produce about 37 μg diazepam metabolites/mg protein;cryopreserved cells produce about 30 μg/mg protein. The immortalizedcell lines of the invention can be frozen at -80° C. or in liquidnitrogen for indefinite periods without loss of activity (TABLE 6).Given the ease of storage and maintenance of activity of theimmortalized hepatocyte, on demand delivery of a medical devicecontaining these cells is possible. LADs which utilize immortalizedhepatocytes present no greater demands for usage than the technologycommon to kidney dialysis programs.

                  TABLE 6    ______________________________________    CELL LINE I                         Diazepam metabolites    Storage    Time (days)                         μg/mg protein    ______________________________________    4°  1         32.88    4°  3         25.90    -80°               14        36.26    -30°               1         22.20    dry ice    1         28.63    dry ice    2         21.05    ______________________________________

Immortalized hepatocytes also form the basis of an improved toxicologyassay. Toxicology testing has typically been performed in vivo. In manycases, the metabolic pathways of the animals used differ considerablyfrom those of the human. Additionally, there is increasing publicconcern over the use of animals for toxicity studies creating a need foralternative methods of drug screening. The hepatocyte cell linesdescribed herein provide an alternative to conventional in vivotoxicology testing.

Other embodiments are within the following claims.

What is claimed is:
 1. A virally-immortalized porcine hepatocyte, saidhepatocyte(a) being derived from a normal liver cell; (b) havingretained differentiated hepatocyte-specific metabolic activity; and (c)having the ability to proliferate, wherein said hepatocyte isnontumorigenic after prolonged culture.
 2. The hepatocyte of claim 1,wherein said metabolic activity comprises P450 enzyme activity.
 3. Thehepatocyte of claim 2, wherein said P450 enzyme activity is increased bycontacting said hepatocyte with a metabolic inducer.
 4. The hepatocyteof claim 1, wherein said hepatocyte comprises substantially pure simianvirus 40 (SV40) DNA.
 5. The hepatocyte of claim 4, wherein said DNAencodes wild type SV40 large T antigen (TAg).
 6. The hepatocyte of claim5, wherein expression of said TAg is constitutive.
 7. The hepatocyte ofclaim 6, wherein said hepatocyte further comprises a substantially puretumor suppressor-encoding DNA.
 8. The hepatocyte of claim 4, whereinsaid DNA encodes a temperature-sensitive TAg.
 9. The hepatocyte of claim8, wherein said hepatocyte further comprises a substantially pure tumorsuppressor-encoding DNA.
 10. The hepatocyte of claim 7 or 9, whereinsaid hepatocyte comprises a substantially pure human p53-encoding DNA.11. The hepatocyte of claim 5, wherein expression of said TAg isinducible.
 12. A method of evaluating the toxicity of a compound invitro, comprising(a) providing the hepatocyte of claim 1; (b) contactingsaid hepatocyte with said compound; and (c) measuring the viability ofsaid hepatocyte, wherein a decrease in viability in the presence of saidcompound compared to that in the absence of said compound indicates thatsaid compound is toxic in vivo.
 13. A method of evaluating the toxicityof a compound in vitro, comprising(a) providing the hepatocyte of claim1; (b) contacting said hepatocyte with said compound; and (c) measuringthe metabolic activity of said hepatocyte, wherein a decrease inmetabolic activity in the presence of said compound compared to that inthe absence of said compound indicates that said compound is toxic invivo.
 14. A method of neutralizing a toxic compound in a bodily fluid ofa mammal, comprising contacting said fluid with the hepatocyte of claim1 in vitro.
 15. A method of evaluating the toxicity of a compound invitro, comprising(a) providing a metabolically-active hepatocyte; (b)contacting said metabolically-active hepatocyte with said compound togenerate a cell supernatant; (c) removing said cell supernatant fromsaid metabolically-active hepatocyte; (d) providing the hepatocyte ofclaim 1; (e) contacting said hepatocyte with said supernatant; and (f)measuring the metabolic activity of said hepatocyte, wherein a decreasein metabolic activity in the presence of said supernatant compared tothat in the absence of said supernatant indicates that said compound istoxic in vivo.
 16. A method of evaluating the toxicity of a compound invitro, comprising(a) providing a metabolically-active hepatocyte; (b)contacting said metabolically-active hepatocyte with said compound togenerate a cell supernatant; (c) removing said cell supernatant fromsaid metabolically-active hepatocyte; (d) providing the hepatocyte ofclaim 1; (e) contacting said hepatocyte with said supernatant; and (f)measuring the viability of said hepatocyte, wherein a decrease inviability in the presence of said supernatant compared to that in theabsence of said supernatant indicates that said compound is toxic invivo.
 17. A method of neutralizing a toxic compound in a bodily fluid ofa mammal, comprising contacting said fluid with the hepatocyte of claim1, wherein said contacting step takes place in a perfusion device. 18.The method of claim 17, wherein said mammal is a human.
 19. The methodof claim 17, wherein said perfusion device is a liver assist device. 20.The method of claim 17, wherein said perfusion device is a bioreactor.21. A perfusion device comprising(a) a housing defining a perfusioninlet and a perfusion outlet; (b) a porous membrane structure mountedwithin said housing to define a perfusion compartment and an adjacenthepatocyte compartment; and (c) virally-immortalized porcinehepatocytes, said hepatocytes (1) being derived from a normal livercell, (2) having differentiated hepatocyte-specific metabolic activity,and (3) having the ability to proliferate, wherein said hepatocyte isnontumorigenic after prolonged culture.
 22. A perfusion devicecomprising(a) a housing defining a perfusion inlet and a perfusionoutlet; (b) a porous membrane structure mounted within said housing todefine a perfusion compartment and an adjacent hepatocyte compartment;and (c) virally-immortalized mammalian hepatocytes, said hepatocytes (1)being derived from a normal liver cell, (2) having differentiatedhepatocyte-specific metabolic activity, (3) comprising substantiallypure DNA encoding wild type SV40 TAg, wherein expression of said TAg isinducible; and (4) having the ability to proliferate, wherein saidhepatocyte is nontumorigenic after prolonged culture.
 23. Avirally-immortalized mammalian hepatocyte, said hepatocyte(a) beingderived from a normal liver cell; (b) having retained differentiatedhepatocyte-specific metabolic activity; (c) comprising substantiallypure DNA encoding wild type SV40 TAg, wherein expression of said TAg isinducible; and (d) having the ability to proliferate, wherein saidhepatocyte is nontumorigenic after prolonged culture.
 24. The hepatocyteof claim 23, wherein said hepatocyte further comprises a substantiallypure tumor suppressor-encoding DNA.
 25. The hepatocyte of claim 24,wherein said DNA encodes human p53.
 26. The hepatocyte of claim 22,wherein said DNA is operably linked to an inducible promoter.
 27. Thehepatocyte of claim 26, wherein said promoter is not derived from SV40.28. The hepatocyte of claim 23, wherein said expression is induced bydexamethasone.
 29. A method of neutralizing a toxic compound in a bodilyfluid of a mammal, comprising contacting said fluid with the hepatocyteof claim 23 in vitro.
 30. The method of claim 29, wherein said mammal isa human.
 31. A method of evaluating the toxicity of a compound in vitro,comprising(a) providing a metabolically-active hepatocyte; (b)contacting said metabolically-active hepatocyte with said compound togenerate a cell supernatant; (c) removing said cell supernatant fromsaid metabolically-active hepatocyte; (d) providing the hepatocyte ofclaim 23; (e) contacting said hepatocyte with said supernatant; and (f)measuring the viability of said hepatocyte, wherein a decrease inviability in the presence of said supernatant compared to that in theabsence of said supernatant indicates that said compound is toxic invivo.
 32. A method of evaluating the toxicity of a compound in vitro,comprising(a) providing the hepatocyte of claim 23; (b) contacting saidhepatocyte with said compound; and (c) measuring the viability of saidhepatocyte, wherein a decrease in viability in the presence of saidcompound compared to that in the absence of said compound indicates thatsaid compound is toxic in vivo.
 33. A method of evaluating the toxicityof a compound in vitro, comprising(a) providing the hepatocyte of claim23; (b) contacting said hepatocyte with said compound; and (c) measuringthe metabolic activity of said hepatocyte, wherein a decrease inmetabolic activity in the presence of said compound compared to that inthe absence of said compound indicates that said compound is toxic invivo.
 34. A method of evaluating the toxicity of a compound in vitro,comprising(a) providing a metabolically-active hepatocyte; (b)contacting said metabolically-active hepatocyte with said compound togenerate a cell supernatant; (c) removing said cell supernatant fromsaid metabolically-active hepatocyte; (d) providing the hepatocyte ofclaim 23; (e) contacting said hepatocyte with said supernatant; and (f)measuring the metabolic activity of said hepatocyte, wherein a decreasein metabolic activity in the presence of said supernatant compared tothat in the absence of said supernatant indicates that said compound istoxic in vivo.
 35. A method of neutralizing a toxic compound in a bodilyfluid of a mammal, comprising contacting said fluid with the hepatocyteof claim 23, wherein said contacting step takes place in a perfusiondevice.
 36. The method of claim 35, wherein said perfusion device is aliver assist device.
 37. The method of claim 35, wherein said mammal isa human.
 38. The method of claim 35, wherein said perfusion device is abioreactor.